Arthropods as Vectors of Grapevine Trunk Disease Pathogens: Quantification of Phaeomoniella chlamydospora on Arthropods and Mycobiome Analysis of Earwig Exoskeletons.

Viticulture worldwide is challenged by grapevine trunk diseases (GTDs). Involvement of arthropods in the dissemination process of GTD pathogens, notably esca pathogens, is indicated after detection of associated pathogens on arthropod exoskeletons, and demonstration of transmission under artificial conditions. The present study is the first to quantify spore loads via qPCR of the esca-relevant pathogen Phaeomoniella chlamydospora on arthropods collected in German vineyards, i.e., European earwigs (Forficula auricularia), ants (Formicidae), and two species of jumping spiders (Marpissa muscosa and Synageles venator). Quantification of spore loads showed acquisition on exoskeletons, but most arthropods carried only low amounts. The mycobiome on earwig exoskeletons was described for the first time to reveal involvement of earwigs in the dispersal of GTDs in general. Metabarcoding data support the potential risk of earwigs as vectors for predominantly Pa. chlamydospora and possibly Eutypa lata (causative agent of Eutypa dieback), as respective operational taxonomical unit (OTU) assigned genera had relative abundances of 6.6% and 2.8% in total reads, even though with great variation between samples. Seven further GTD-related genera were present at a very low level. As various factors influence the successful transmission of GTD pathogens, we hypothesize that arthropods might irregularly act as direct vectors. Our results highlight the importance of minimizing and protecting pruning wounds in the field.

Molecular identification of endophytes from maize roots and their biocontrol potential against toxigenic fungi of Nigerian maize.

Plants benefit from plant-associated microorganisms, of which endophytes are of particular interest as they are transmitted from generation to generation. This study characterises endophytes from maize roots and determines their biocontrol potential against toxigenic fungi in Nigerian maize. Maize roots were collected from farms in Lafia, and stored grain samples were collected from the six Northern States of Nigeria, from which endophytes and toxigenic fungal strains were isolated. Molecular identification employing 16SrRNA/internal transcribed spacer (ITS) sequences for isolated fungal endophytes was carried out, and mycotoxins produced by fungi were determined by high-performance liquid chromatography analysis. Biocontrol activity of the endophytes was determined using the dual culture confrontation test. Aspergillus and Fusarium genera were the prevalent isolated fungal species. Eight fungal endophytes were identified of which Trichoderma harzianum, Dichotomopilus erectus and Burkholderia spp. were the isolates with biocontrol activities, while 12 Aspergillus spp. were found to produce varying amounts of ochratoxin A and aflatoxin B1, respectively. T. harzianum showed the best inhibition (74%), followed by D. erectus (50%) and Burkholderia spp. (48%). T. harzianum showed poor inhibition of Aspergillus flavus (B7) at 30%. However, results from the Pakdaman Biological Control Index showed that T. harzianum has the best antifungal biocontrol activity of the three endophytes. The study concludes that antifungal biocontrol agents can be sourced from endophytes to obtain indigenous control activities that can check mycotoxin contamination of food and livestock feed, as well as elucidate possible metabolites for agricultural and industrial applications, which will help improve plant performance, increase crop yield and sustainability.

Diagnosis of Soybean Diseases Caused by Fungal and Oomycete Pathogens: Existing Methods and New Developments.

Soybean (Glycine max) acreage is increasing dramatically, together with the use of soybean as a source of vegetable protein and oil. However, soybean production is affected by several diseases, especially diseases caused by fungal seed-borne pathogens. As infected seeds often appear symptomless, diagnosis by applying accurate detection techniques is essential to prevent propagation of pathogens. Seed incubation on culture media is the traditional method to detect such pathogens. This method is simple, but fungi have to develop axenically and expert mycologists are required for species identification. Even experts may not be able to provide reliable type level identification because of close similarities between species. Other pathogens are soil-borne. Here, traditional methods for detection and identification pose even greater problems. Recently, molecular methods, based on analyzing DNA, have been developed for sensitive and specific identification. Here, we provide an overview of available molecular assays to identify species of the genera Diaporthe, Sclerotinia, Colletotrichum, Fusarium, Cercospora, Septoria, Macrophomina, Phialophora, Rhizoctonia, Phakopsora, Phytophthora, and Pythium, causing soybean diseases. We also describe the basic steps in establishing PCR-based detection methods, and we discuss potentials and challenges in using such assays.

Major proliferation of transposable elements shaped the genome of the soybean rust pathogen Phakopsora pachyrhizi.

With >7000 species the order of rust fungi has a disproportionately large impact on agriculture, horticulture, forestry and foreign ecosystems. The infectious spores are typically dikaryotic, a feature unique to fungi in which two haploid nuclei reside in the same cell. A key example is Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease, one of the world's most economically damaging agricultural diseases. Despite P. pachyrhizi's impact, the exceptional size and complexity of its genome prevented generation of an accurate genome assembly. Here, we sequence three independent P. pachyrhizi genomes and uncover a genome up to 1.25 Gb comprising two haplotypes with a transposable element (TE) content of ~93%. We study the incursion and dominant impact of these TEs on the genome and show how they have a key impact on various processes such as host range adaptation, stress responses and genetic plasticity.

Successful Silencing of the Mycotoxin Synthesis Gene TRI5 in Fusarium culmorum and Observation of Reduced Virulence in VIGS and SIGS Experiments.

Crops constantly experience various biotic stresses during their life cycle, and Fusarium spp. remain one of the most serious groups of pathogens affecting plants. The ability to manipulate the expression of certain microorganism genes via RNAi creates the opportunity for new-generation dsRNA-based preparations to control a large number of diseases. In this study, we applied virus-induced gene silencing (VIGS), and spray-induced gene silencing (SIGS) to silence the trichothecene-producing gene TRI5 in F. culmorum as a means to reduce its aggressiveness on spring wheat. Treatment of the fungus with dsTRI5RNA in vitro reduced deoxynivalenol (DON) and 3-acetyldeoxynivalenol (3-A-DON) accumulations by 53-85% and 61-87%, respectively, and reduced TRI5 expression by 84-97%. VIGS decreased the proportion of infected wheat spikelets by 73%, but upregulation was observed for TRI5. SIGS on wheat leaves and ears using certain dsTRI5RNA amounts negatively impacted F. culmorum growth. However, when performing in vivo analyses of TRI5 mRNA levels, the upregulation of the gene was determined in the variants where fungal colonization was restricted, suggesting a compensatory reaction of the pathogen to RNAi.

Identification and Characterization of Nep1-Like Proteins From the Grapevine Downy Mildew Pathogen Plasmopara viticola.

The obligate biotrophic oomycete Plasmopara viticola causes tremendous problems in viticulture by evoking grapevine downy mildew. P. viticola, like other plant pathogens, achieves infection by suppression of plant innate immunity by secretion of effector molecules into its host plant. An ever-expanding family of proteins with effector-like characteristics is formed by the "Necrosis and Ethylene inducing peptide 1 (Nep1)-like proteins" (NLPs). NLPs can be divided into two groups by their ability to induce necrosis. While cytotoxic NLPs may act as virulence factors for a necrotrophic or hemibiotrophic plant pathogen, the role of non-cytotoxic NLPs is so far unknown. In this study, we identified eight independent NLPs in P. viticola and selected three for functional analysis. While one was identified as a putative pseudo gene, two contain all so far described critical key elements for necrosis formation except for an N-terminal signal peptide. Further characterization revealed that none of the putative necrosis elicitors was able to actually induce necrosis, neither in several susceptible or resistant Vitis species nor in the dicot model plant Nicotiana benthamiana. This inability exists independently of the presence or absence of a signal peptide. However, any possible mechanism for the suppression of the ability to induce necrosis in planta was not detected. Interestingly, expression analysis of the presumed pseudo gene revealed remarkable differences between pure sporangia solution and sporangia in the presence of leaf material. To our knowledge, this is the first report of this kind of regulation that suggests an important function of so far nonfunctional "pseudo" NLP genes during the first hours of infection.

Host-Induced Gene Silencing of the MAPKK Gene PsFUZ7 Confers Stable Resistance to Wheat Stripe Rust.

RNA interference (RNAi) is a powerful genetic tool to accelerate research in plant biotechnology and control biotic stresses by manipulating target gene expression. However, the potential of RNAi in wheat to efficiently and durably control the devastating stripe rust fungus Puccinia striiformis f. sp. tritici (Pst) remained largely under explored so far. To address this issue, we generated transgenic wheat (Triticum aestivum) lines expressing dsRNA targeting PsFUZ7 transcripts of Pst We analyzed expression of PsFUZ7 and related genes, and resistance traits of the transgenic wheat lines. We show that PsFUZ7 is an important pathogenicity factor that regulates infection and development of Pst A PsFUZ7 RNAi construct stably expressed in two independent transgenic wheat lines confers strong resistance to PstPst hyphal development is strongly restricted, and necrosis of plant cells in resistance responses was significantly induced. We conclude that trafficking of RNA molecules from wheat plants to Pst may lead to a complex molecular dialogue between wheat and the rust pathogen. Moreover, we confirm the RNAi-based crop protection approaches can be used, to our knowledge, as a novel control strategy against rust pathogens in wheat.

Basidiomycete-specific PsCaMKL1 encoding a CaMK-like protein kinase is required for full virulence of Puccinia striiformis f. sp. tritici.

Calcium/calmodulin-dependent kinases (CaMKs) are Ser/Thr protein kinases (PKs) that respond to changes in cytosolic free Ca2+ and play diverse roles in eukaryotes. In fungi, CAMKs are generally classified into four families CAMK1, CAMKL, RAD53 and CAMK-Unique. Among these, CAMKL constitutes the largest family. In some fungal plant pathogens, members of the CaMKL family have been shown to be responsible for pathogenesis. However, little is known about their role(s) in rust fungi. In this study, we functionally characterized a novel PK gene, PsCaMKL1, from Puccinia striiformis f. sp. tritici (Pst). PsCaMKL1 belongs to a group of PKs that is evolutionarily specific to basidiomyceteous fungi. PsCaMKL1 shows little intra-species polymorphism between Pst isolates. PsCaMKL1 transcripts are highly elevated at early infection stages, whereas gene expression is downregulated in barely germinated urediospores under KN93 treatment. Overexpression of PsCaMKL1 in fission yeast increased resistance to environmental stresses. Knock down of PsCaMKL1 using host-induced gene silencing (HIGS) reduced the virulence of Pst accompanied by reactive oxygen species (ROS) accumulation and a hypersensitive response. These results suggest that PsCaMKL1 is a novel pathogenicity factor that exerts it virulence function by regulating ROS production in wheat.

Gα proteins Gvm2 and Gvm3 regulate vegetative growth, asexual development, and pathogenicityon apple in Valsa mali.

In fungi, heterotrimeric guanine-nucleotide binding proteins (G-proteins) are key elements of signal transduction pathways, which control growth, asexual and sexual development, as well as virulence. In this study, we have identified two genes encoding heterotrimeric G protein alpha subunits, named Gvm2 and Gvm3, from Valsa mali, the causal agent of apple Valsa canker. Characterization of Gvm2 and Gvm3 mutants indicates that Gvm3 may be a crucial regulator of vegetative growth. Deletion of the corresponding gene results in a 20% reduction in growth rate. Besides, Gvm2 and Gvm3 seem to be involved in asexual reproduction, and mutants are hypersensitive to oxidative and cell membrane stresses. Interestingly, both G protein alpha subunits were most probably involved in V. mali virulence. In infection assays using Malus domestica cv. 'Fuji' leaves and twigs, the size of lesions caused by deletion mutants △Gvm2, or △Gvm3 are significantly reduced. Furthermore, many genes encoding hydrolytic enzymes-important virulence factors in V. mali-are expressed at a lower level in these deletion mutants. Our results suggest that Gvm2 and Gvm3 play an important role in virulence probably by regulation of expression of cell wall degrading enzymes. △Gvm2, and △Gvm3 mutants were further analyzed with respect to their impact on the transcript levels of genes in the cAMP/PKA pathway. The expression of the genes encoding adenylate cyclase VmAC, protein kinase A (PKA) regulatory subunit VmPKR, and PKA catalytic subunit VmPKA1 are down-regulated in both mutants. Further analyses indicated that intracellular cAMP level and PKA activity are down-regulated in the △Gvm3 mutant, but are basically unchanged in the △Gvm2 mutant. Overall, our findings indicate that both Gvm2 and Gvm3 play diverse roles in the modulation of vegetative growth, asexual development, and virulence in V. mali.

Agrobacterium tumefaciens-mediated transformation of the causative agent of Valsa canker of apple tree Valsa mali var. mali.

Valsa mali var. mali (Vmm), which is the causative agent of Valsa canker of apple tree, causes heavy damage to apple production in eastern Asia. In this article, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of Vmm and expression of gfp (green fluorescent protein) in this fungus. The transformation system was optimized to a transformation efficiency of approximately 150 transformants/10(6) conidia, and a library containing over 4,000 transformants was generated. The tested transformants were mitotically stable. One hundred percent hph (hygromycin B phosphotransferase) integration into Vmm was identified by PCR and five single-copy integration of T-DNA was detected in the eighteen transformants by Southern blot. To our knowledge, this is the first report of ATMT of Vmm. Furthermore, this library has been used to identify genes involved in the virulence of the pathogen, and the transformation system may also be useful to the transformation of other species of the genus Valsa.